Regulation of ryanodine receptors in the heart.

In response to exercise or other stresses, catecholamines are released into the circulation and within the heart. Catecholaminergic stimulation of 1-adrenergic receptors ( 1ARs) in the heart increases heart rate (chronotropy) and contractility (inotropy), resulting in increased cardiac output during acute stress. Concurrent stimulation of 2-ARs dilates blood vessels, which increase blood flow to exercising muscles, thereby matching increased cardiac output to metabolic demands of the organs. The inotropic mechanisms investigated by Valdivia and colleagues1 in the current issue of Circulation Research are essential for the acute stress-dependent increase of cardiac output. Each of the millions of muscle cells in the heart (cardiomyocytes) contribute to myocardial force development. Cardiomyocyte contraction is controlled by intracellular Ca release through a process called excitationcontraction coupling (ECC) that involves the following steps: (1) an action potential (AP) depolarizes the cell membrane; (2) voltage-dependent plasma membrane L-type calcium channels (Cav1.2) opening results in a whole-cell inward Ca current (ICa); (3) ICa activates cardiac ryanodine receptor (RyR2)/Ca release channels located on the junctional sarcoplasmic reticulum (jSR), a process referred to as Ca induced Ca release (CICR); (4) Ca binds to troponin C (TnC) leading to cross-bridge formation between myosin and actin and contraction of the sarcomere. Cardiomyocyte relaxation is signaled by a return of intracellular [Ca ] to resting levels attributable to the following major mechanisms: (1) Cav1.2 inactivation; (2) RyR2 inactivation; (3) Ca reuptake into the SR by SERCA2a pumps; and (4) Na /Ca exchange extrusion of Ca to the extracellular space. Under resting conditions, net SR Ca release contributes approximately 66% of Ca necessary for myofilament activation in large mammals including humans, and approximately 90% in rodents.2 Stimulation of -ARs in the heart results in cAMPmediated activation of protein kinase A (PKA), which phosphorylates multiple intracellular proteins including Cav1.2, RyR2,4 and the SERCA2a inhibitor phospholamban (PLB).5 PLB phosphorylation at Ser16 by PKA dissociates PLB from SERCA2a and increases SR calcium uptake.5 Cav1.2 -subunit phosphorylation at Ser1928 increases ICa amplitude and shifts activation threshold to more negative voltages.6 Using intact hearts or cardiomyocytes, multiple studies have found that RyR2 is phosphorylated by PKA during -AR stimulation,7–15 and that PKA phosphorylation increases the single-channel open probability.4,7,8,16–18 As RyR2 mediates CICR, these observations suggest that PKA-mediated RyR2 phosphorylation may directly contribute to increased cardiac function during -adrenergic stimulation (ie, during stress). Despite much evidence, the fundamental question of whether RyR2 PKA phosphorylation by itself increases SR Ca release independent from ICa and SR Ca load has been the subject of ongoing debate. Characterizing the RyR2-specific component of SR Ca release in intact cells during -adrenergic stimulation has been a major challenge. Both SR Ca load and ICa have been recognized as important mediators of SR Ca release during inotropic modulation. ICa and [Ca ]i can be directly measured with patch-clamp and fluorescence indicators, respectively. Therefore activation of RyR2 during CICR in intact cells has been assessed by measuring intracellular Ca release as a function of voltage-dependent ICa. Thus, the “normalized” increase of [Ca ]i/ICa, referred to as ECC “gain”, can be used to dissect the catecholamine-dependent role of RyR2 in CICR. Previously, isolation of PKA effects on RyR2 function has been addressed indirectly using pharmacological modulators and [Ca ]SR loading protocols in cardiomyocytes. For example, using micromolar caffeine application in paced cardiomyocytes, Eisner and colleagues observed that the amplitude of intracellular [Ca ]i release increased only transiently leading them to conclude that RyR2 modulation has no maintained effect on CICR gain.21 Bers and colleagues reported that catecholamine stimulation failed to increase the CICR gain in cells examined under conditions of pacingcontrolled SR load and ICa. However, they did note that the maximal rate of CICR was significantly faster during -AR stimulation indicating potentially important functional effects of RyR2 PKA phosphorylation.22 Lederer and colleagues showed that PLB KO cardiomyocytes had increased SERCA2a activity and displayed constitutively increased SR Ca load and CICR gain indicating modulation of RyR2 and inotropic function by SR Ca load.23 Bers and colleagues have used saponin-permeabilized PLB KO cardiomyocytes and found that cAMP could not increase Ca spark rate (however they performed their experiments at subphysiological Ca concentrations, conditions under which the RyR2 channels are known to be tightly closed), which was interpreted as evidence against a role of RyR2 for inotropic modulation.24 In contrast, Lindegger and Niggli studied subcellular mechanisms of CICR using 2-photon limited The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Clyde and Helen Wu Center for Molecular Cardiology, Departments of Physiology and Cellular Biophysics (S.E.L., A.R.M.) and Medicine (A.R.M.), College of Physicians and Surgeons of Columbia University, New York. Correspondence to Andrew R. Marks, the Clyde and Helen Wu Center for Molecular Cardiology, Departments of Physiology and Cellular Biophysics and Medicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032. E-mail [email protected] (Circ Res. 2007;101:746-749.) © 2007 American Heart Association, Inc.